Treating sickle cell disease with a pyruvate kinase r activating compound

ABSTRACT

Compounds that activate pyruvate kinase R can be used for the treatment of sickle cell disease (SCD). Methods and compositions for the treatment of SCD are provided herein, including a therapeutic compound designated as Compound 1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/576,720, filed on Sep. 19, 2019, which claims the benefit of U.S.Provisional Application No. 62/733,558, filed on Sep. 19, 2018, U.S.Provisional Application No. 62/733,562, filed on Sep. 19, 2018, U.S.Provisional Application No. 62/782,933, filed on Dec. 20, 2018, U.S.Provisional Application No. 62/789,641, filed on Jan. 8, 2019, and U.S.Provisional Application No. 62/811,904, filed on Feb. 28, 2019, each ofwhich is incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to the treatment of sickle cell disease (SCD),including the treatment of patients diagnosed with SCD by theadministration of a compound that activates pyruvate kinase R (PKR).

BACKGROUND

Sickle cell disease (SCD) is a chronic hemolytic anemia caused byinheritance of a mutated form of hemoglobin (Hgb), sickle Hgb (HgbS). Itis the most common inherited hemolytic anemia, affecting 70,000 to80,000 patients in the United States (US). SCD is characterized bypolymerization of HgbS in red blood cells (RBCs) when HgbS is in thedeoxygenated state (deoxy-HgbS), resulting in a sickle-shapeddeformation. Sickled cells aggregate in capillaries precipitatingvaso-occlusive events that generally present as acute and painful crisesresulting in tissue ischemia, infarction, and long-term tissue damage.RBCs in patients with SCD tend to be fragile due to sickling and otherfactors, and the mechanical trauma of circulation causes hemolysis andchronic anemia. Finally, damaged RBCs have abnormal surfaces that adhereto and damage vascular endothelium, provoking aproliferative/inflammatory response that underlies large-vessel strokeand potentially pulmonary-artery hypertension. Collectively, thesecontribute to the significant morbidity and increased mortalityassociated with this disease.

Currently, therapeutic treatment of SCD is inadequate. The only knowncure for SCD is hematopoietic stem cell transplantation which hasserious risks, is typically recommended for only the most serious cases,and is largely offered only to children with sibling-matched donors.Gene therapy is also under investigation with promising preliminaryresults; however, there are market access hurdles, mainly high cost andtreatment complexities, that are likely to limit its broad use in thenear term. There have been few advances in therapies for SCD over thepast two decades. Hydroxyurea (HU) induces HgbF which interrupts thepolymerization of HgbS, and thereby has activity in decreasing the onsetof vaso-occlusive crises and pathological sequelae of SCD. While HU isin wide use as a backbone therapy for SCD, it remains only partiallyeffective, and is associated with toxicity, such as myelosuppression andteratogenicity. Patients receiving HU still experience hemolysis,anemia, and vaso-occlusive crises, suggesting a need for more effectivetherapies, either as a replacement or in combination with HU. Beyond HU,therapeutic intervention is largely supportive care, aimed at managingthe symptoms of SCD. For instance, blood transfusions help with theanemia and other SCD complications by increasing the number of normalRBCs. However, repeated transfusions lead to iron overload and the needfor chelation therapies to avoid consequent tissue damage. In additionto these approaches, analgesic medications are used to manage pain.

Given the current standard of care for SCD, there is a clear medicalneed for a noninvasive, disease-modifying therapy with appropriatesafety and efficacy profiles.

SUMMARY

One aspect of the disclosure relates to methods of treating SCDcomprising the administration of a therapeutically effective amount of apyruvate kinase R (PKR) activator to a patient in need thereof diagnosedwith SCD. Pyruvate kinase R (PKR) is the isoform of pyruvate kinaseexpressed in RBCs, and is a key enzyme in glycolysis. The invention isbased in part on the discovery that the activation of PKR can targetboth sickling, by reducing deoxy-HgbS, and hemolysis. Targetinghemolysis may be achieved by improving RBC membrane integrity. Oneaspect of the disclosure is the recognition that activation of PKR canreduce 2,3-diphosphoglycerate (2,3-DPG), which leads to decreaseddeoxy-HgbS (and, therefore, sickling), as well as can increase ATP,which promotes membrane health and reduces hemolysis. Another aspect ofthe disclosure is the recognition that activation of PKR can reduce2,3-diphosphoglycerate (2,3-DPG), which inhibits Hgbdeoxygenation/increases oxygen affinity of HgbS and leads to decreaseddeoxy-HgbS (and, therefore, sickling), as well as can increase ATP,which promotes membrane health and reduces hemolysis. Accordingly, inone embodiment, PKR activation (e.g., by administration of atherapeutically effective amount of a PKR Activating Compound to apatient diagnosed with SCD) reduces RBC sickling via a reduction inlevels of 2,3-diphosphoglycerate (2,3-DPG), which in turn reduces thepolymerization of sickle Hgb (HgbS) into rigid aggregates that deformthe cell. Furthermore, in some embodiments, PKR activation maycontribute to overall RBC membrane integrity via increasing levels ofadenosine triphosphate (ATP), which is predicted to reducevaso-occlusive and hemolytic events which cause acute pain crises andanemia in SCD patients.

Preferably, a patient diagnosed with SCD is treated with a compound thatis a PKR Activating Compound. The PKR activator can be a compoundidentified as a PKR Activating Compound or a composition identified as aPKR Activating Composition having an AC₅₀ value of less than 1 μM usingthe Luminescence Assay described in Example 2, or a pharmaceuticallyacceptable salt and/or other solid form thereof. For example, the PKRActivating Compound can be the compound(S)-1-(5-((2,3-dihydro[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one(Compound 1):

or a pharmaceutically acceptable salt thereof. Compound 1 is aselective, orally bioavailable PKR Activating Compound that decreases2,3-DPG, increases ATP, and has anti-sickling effects in disease modelswith a wide therapeutic margin relative to preclinical toxicity.

PKR Activating Compounds can be readily identified as compounds ofFormula I:

or a pharmaceutically acceptable salt thereof, (e.g., Compound 1 andmixtures of Compound 1 with its stereoisomer) having an AC₅₀ value ofless than 1 μM using the Luminescence Assay described in Example 2.

In other embodiments, the PKR Activating Compound can be any of thecompounds listed in FIG. 1, or a pharmaceutically acceptable saltthereof.

PKR Activating Compounds, such as1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, or apharmaceutically acceptable salt thereof, are useful in pharmaceuticalcompositions for the treatment of patients diagnosed with SCD. PKRActivating Compounds, such as any of the compounds listed in FIG. 1, ora pharmaceutically acceptable salt thereof, are useful in pharmaceuticalcompositions for the treatment of patients diagnosed with SCD. Thecompositions comprising a compound of Formula I (e.g., Compound 1), or apharmaceutically acceptable salt thereof, can be obtained by certainprocesses also provided herein. The compositions comprising any of thecompounds listed in FIG. 1, or a pharmaceutically acceptable saltthereof, can be obtained by certain processes also provided herein.

The methods of treating SCD provided herein can offer greater protectionagainst vaso-occlusive crises and hemolytic anemia, as compared toexisting and emerging therapies. Therefore, use of a PKR ActivatingCompound, such as Compound 1, provides a novel and improved therapeuticapproach either alone or in combination with drugs that act throughalternative mechanisms, such as hydroxyurea (HU). In addition, use of aPKR Activating Compound, such as any of the compounds listed in FIG. 1,provides a novel and improved therapeutic approach either alone or incombination with drugs that act through alternative mechanisms, such ashydroxyurea (HU).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table of PKR Activating Compounds.

FIG. 2 is a schematic showing the relationship of PKR activation to thereduction of the clinical consequences of sickle cell disease (SCD).

FIG. 3 is a graph showing the oxyhemoglobin dissociation curve andmodulating factors by plotting the relationship between hemoglobinsaturation (percent) vs. partial pressure of oxygen (mmHg).

FIG. 4A is a chemical synthesis scheme for compounds of Formula I,including a synthesis of Compound 1 (separately provided in FIG. 4B).

FIG. 4B is a chemical synthesis scheme for Compound 1.

FIG. 4C is a general chemical synthesis of the compounds listed in FIG.1.

FIG. 5 is a graph showing activation of recombinant PKR-R510Q withCompound 1, plotting the normalized rate vs. concentration ofphosphoenolpyruvate (PEP) (Example 3).

FIG. 6 is a graph of data showing activation of recombinant PKR-R510Q byCompound 1 in the enzyme assay of Example 3.

FIG. 7 is a graph of data showing PKR activation in human red bloodcells treated with Compound 1 (Example 4).

FIG. 8A (Study 1) and FIG. 8B (Study 2) are each graphs showing theobserved changes in 2,3-DPG levels in blood from mice following 7 daysof once daily (QD) oral treatment with Compound 1 (Example 5).

FIG. 9 is a graph showing observed changes in 2,3-DPG levels in bloodfrom mice following 7 days of once daily (QD) oral treatment withCompound 1 (Example 5, Study 2).

FIG. 10A (Study 1) and FIG. 10B (Study 2) are graphs of data measuringATP concentrations in red blood cells of mice following 7 days of oncedaily (QD) oral treatment with Compound 1 (Example 5).

FIG. 11 is a graph of the blood 2,3-DPG levels measured over time inhealthy volunteers who received a single dose of Compound 1 or placebo.

FIG. 12 is a graph of the blood 2,3-DPG levels measured 24 hourspost-dose in healthy volunteers who received a single dose of Compound 1or placebo.

FIG. 13 is a graph of the blood 2,3-DPG levels measured over time inhealthy volunteers who received daily doses of Compound 1 or placebo for14 days.

FIG. 14 is a graph of the blood 2,3-DPG levels measured on day 14 inhealthy volunteers who received daily doses of Compound 1 or placebo for14 days.

FIG. 15 is a graph of the blood ATP levels measured on day 14 in healthyvolunteers who received daily doses of Compound 1 or placebo for 14days.

FIG. 16 is a graph plotting the blood concentration of Compound 1(ng/mL) measured in healthy volunteer (HV) patients on a first (left)axis and the concentration of 2,3-DPG (micrograms/mL) measured in theseHV patients on a second (right) axis after administration of a singledose of Compound 1 (400 mg).

DETAILED DESCRIPTION

Methods of treating SCD preferably include administration of atherapeutically effective amount of a compound (e.g., Compound 1) thatreduces HgbS polymerization, for example by increasing HgbS affinity foroxygen. Methods of treating SCD also preferably include administrationof a therapeutically effective amount of a compound (e.g., any of thecompounds listed in FIG. 1) that reduces HgbS polymerization, forexample by increasing HgbS affinity for oxygen. Methods of lowering2,3-DPG and/or increasing ATP levels in human RBCs compriseadministering a PKR Activating Compound, such as Compound 1. Methods oflowering 2,3-DPG and/or increasing ATP levels in human RBCs alsocomprise administering a PKR Activating Compound, such as any of thecompounds listed in FIG. 1. Together these effects are consistent withproviding therapies to reduce HgbS sickling and to improve RBC membranehealth, presenting a unique disease-modifying mechanism for treatingSCD.

A PKR Activating Compound, such as Compound 1, is useful to promoteactivity in the glycolytic pathway. A PKR Activating Compound, such asany of the compounds listed in FIG. 1, also is useful to promoteactivity in the glycolytic pathway. As the enzyme that catalyzes thelast step of glycolysis, PKR directly impacts the metabolic health andprimary functions of RBCs. PKR Activating Compounds (e.g., Compound 1),are useful to decrease 2,3-DPG and increase ATP. PKR ActivatingCompounds (e.g., any of the compounds listed in FIG. 1), are useful todecrease 2,3-DPG and increase ATP. PKR Activating Compounds (e.g.,Compound 1 or any of the compounds listed in FIG. 1, preferablyCompound 1) are also useful to increase Hgb oxygen affinity in RBC. Thedisclosure is based in part on the discovery that PKR activation is atherapeutic modality for SCD, whereby HgbS polymerization and RBCsickling are reduced via decreased 2,3-DPG and increased ATP levels.

SCD is the most common inherited blood disorder and clinically manifestswith potentially severe pathological conditions associated withsubstantial physical, emotional, and economic burden. For instance,acute vaso-occlusive pain crises can be debilitating and necessitaterapid medical response. Chronic hemolytic anemia causes fatigue andoften necessitates blood transfusions and supportive care. Over time,impaired oxygen transport through microvasculature precipitates organand tissue damage. While there are a number of options available fortreating symptoms, overall disease management would benefit fromtherapies that target upstream processes to prevent vaso-occlusion andhemolysis.

The described clinical symptoms are largely due to perturbations in RBCmembrane shape and function resulting from aggregation of HgbSmolecules. Unlike normal Hgb, HgbS polymerizes when it is in thedeoxygenated state and ultimately causes a deformed, rigid membrane thatis unable to pass through small blood vessels, thereby blocking normalblood flow through microvasculature. The loss of membrane elasticityalso increases hemolysis, reducing RBC longevity. Furthermore, decreasedcellular ATP and oxidative damage contribute to a sickle RBC membranethat is stiffer and weaker than that of normal RBCs. The damagedmembrane has a greater propensity for adhering to vasculature, leadingto hemolysis, increased aggregation of sickled RBCs, and increasedcoagulation and inflammation associated with vaso-occlusive crises.

The underlying cause of sickling is the formation of rigid deoxy-HgbSaggregates that alter the cell shape and consequently impact cellularphysiology and membrane elasticity. These aggregates are highlystructured polymers of deoxygenated HgbS; the oxygenated form does notpolymerize. Polymerization is promoted by a subtle shift in conformationfrom the oxygen-bound relaxed (R)-state to the unbound tense (T)-state.In the latter, certain residues within the (3-chain of HgbS are able tointeract in a specific and repetitive manner, facilitatingpolymerization.

The concentration of deoxy-HgbS depends on several factors, but thepredominant factor is the partial pressure of oxygen (PO₂). Oxygenreversibly binds to the heme portions of the Hgb molecule. As oxygenatedblood flows via capillaries to peripheral tissues and organs that areactively consuming oxygen, PO₂ drops and Hgb releases oxygen. Thebinding of oxygen to Hgb is cooperative and the relationship to PO₂levels fits a sigmoidal curve (FIG. 3). This relationship can beimpacted by temperature, pH, carbon dioxide, and the glycolyticintermediate 2,3-DPG. 2,3-DPG binds within the central cavity of the Hgbtetramer, causes allosteric changes, and reduces Hgb's affinity foroxygen. Therefore, therapeutic approaches that increase oxygen affinity(i.e., reduce deoxygenation) of HgbS would presumably decrease polymerformation, changes to the cell membrane, and clinical consequencesassociated with SCD.

One aspect of this disclosure is targeting PKR activation to reduce2,3-DPG levels, based on PKR's role in controlling the rate ofglycolysis in RBCs. A decrease in 2,3-DPG with PKR activation has beendemonstrated in preclinical studies and in healthy volunteers andpatients with pyruvate kinase deficiency. Additionally, PKR activationwould be expected to increase ATP, and has been observed to do so inthese same studies. Given the role of ATP in the maintenance of ahealthy RBC membrane and protection from oxidative stress, elevating itslevels is likely to have broad beneficial effects. Therefore, activationof PKR offers the potential for a 2,3-DPG effect (i.e., reduced cellmembrane damage from HgbS polymerization) that is augmented by ATPsupport for membrane integrity. It is via these changes that a PKRactivator is could positively impact physiological changes that lead tothe clinical pathologies of SCD (FIG. 2). In another aspect, thedisclosure relates to a method of improving the anemia and thecomplications associated with anemia in SCD patients (e.g., ≥12 years ofage) with Hgb SS or Hgb SB⁰-thalassemia.

Compound 1 is a selective, orally bioavailable PKR activator that hasbeen shown to decrease 2,3-DPG, increase ATP, and have anti-sicklingeffects in disease models with a wide therapeutic margin relative topreclinical toxicity.

Methods of treatment can comprise administering to a subject in needthereof a therapeutically effective amount of (i) a PKR ActivatingCompound (e.g., a compound disclosed herein), or a pharmaceuticallyacceptable salt thereof; or (ii) a PKR Activating Composition (e.g., apharmaceutical composition comprising a compound disclosed herein, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier). The pharmaceutical composition may be orallyadministered in any orally acceptable dosage form. In some embodiments,to increase the lifetime of red blood cells, a compound, composition, orpharmaceutical composition described herein is added directly to wholeblood or packed cells extracorporeally or provided to the subject (e.g.,the patient) directly. A patient and/or subject can be selected fortreatment using a compound described herein by first evaluating thepatient and/or subject to determine whether the subject is in need ofactivation of PKR, and if the subject is determined to be in need ofactivation of PKR, then administering to the subject a PKR ActivatingCompound in a pharmaceutically acceptable composition. A patient and/orsubject can be selected for treatment using a compound described hereinby first evaluating the patient and/or subject to determine whether thesubject is diagnosed with SCD, and if the subject is diagnosed with SCD,then administering to the subject a PKR Activating Compound in apharmaceutically acceptable composition. For example, administration ofa therapeutically effective amount of a PKR Activating Compound caninclude administration of a total of about 25 mg-1,500 mg of Compound 1each day, in single or divided doses. In some embodiments, Compound 1 isadministered to patients diagnosed with SCD in total once daily (QD)doses of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, and/or higher iftolerated (e.g., 250 mg, 300 mg, 500 mg, 600 mg, 1000 mg, and/or 1500mg). In some embodiments, a human dose of 80 to 130 mg of Compound 1 isadministered once daily (QD) to a patient in need thereof (e.g., apatient diagnosed with SCD). In some embodiments, a PKR ActivatingCompound is administered in an amount of 400 mg per day (e.g., 400 mg QDor 200 mg BID). In some embodiments, Compound 1 or a pharmaceuticallyacceptable salt thereof is administered in an amount of 400 mg per day(e.g., 400 mg QD or 200 mg BID). In some embodiments, any of thecompounds listed in FIG. 1 or a pharmaceutically acceptable salt thereofis administered in an amount of 400 mg per day (e.g., 400 mg QD or 200mg BID). In some embodiments, a PKR Activating Compound is administeredin an amount of 700 mg per day (e.g., 700 mg QD or 350 mg BID). In someembodiments, Compound 1 or a pharmaceutically acceptable salt thereof isadministered in an amount of 700 mg per day (e.g., 700 mg QD or 350 mgBID). In some embodiments, any of the compounds listed in FIG. 1 or apharmaceutically acceptable salt thereof is administered in an amount of700 mg per day (e.g., 700 mg QD or 350 mg BID). In some embodiments, aPKR Activating Compound is administered in an amount of 100 mg, 200 mg,400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day, in single ordivided doses. In some embodiments, Compound 1 or a pharmaceuticallyacceptable salt thereof is administered in an amount of 100 mg, 200 mg,400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day, in single ordivided doses. In some embodiments, any of the compounds listed in FIG.1 or a pharmaceutically acceptable salt thereof is administered in anamount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mgper day, in single or divided doses.

Methods of treating a patient diagnosed with SCD can includeadministering to the patient in need thereof a therapeutic compoundtargeting reduction of deoxy-HgbS, which may or may not directly improveRBC membrane integrity. Compound 1 has been shown to decrease 2,3-DPGand increase ATP, and reduced cell sickling has been demonstrated indisease models. Accordingly, in some embodiments, the methods oftreatment can address not only sickling, but also hemolysis and anemia.

Methods of treating a patient diagnosed with sickle cell disease, andPKR Activating Compounds for use in such methods, can includeadministering to the patient the PKR Activating Compound (e.g., acomposition comprising one or more compounds of Formula I, such asCompound 1 or a mixture of Compound 1 and Compound 2) in an amountsufficient to reduce 2,3-DPG levels in the patient's red blood cells. Insome embodiments, the amount is sufficient to reduce 2,3-DPG levels byat least 30% after 24 hours, or greater (e.g., reducing 2,3-DPG levelsin the patient's red blood cells by at least 40% after 24 hours). Insome embodiments, the amount is sufficient to reduce 2,3-DPG levels by30-50% after 24 hours. In some embodiments, the amount is sufficient toreduce 2,3-DPG levels by 40-50% after 24 hours. In some embodiments, theamount is sufficient to reduce 2,3-DPG levels by at least 25% after 12hours. In some embodiments, the amount is sufficient to reduce 2,3-DPGlevels by 25-45% after 12 hours. In some embodiments, the amount issufficient to reduce 2,3-DPG levels by at least 15% after 6 hours. Insome embodiments, the amount is sufficient to reduce 2,3-DPG levels by15-30% after 6 hours. In some embodiments, the amount is sufficient toreduce 2,3-DPG levels by at least 40% on day 14 of treatment. In someembodiments, the amount is sufficient to reduce 2,3-DPG levels by 40-60%on day 14 of treatment. In some embodiments, the amount is sufficient toreduce 2,3-DPG levels by at least 50% on day 14 of treatment. In someembodiments, the amount is sufficient to reduce 2,3-DPG levels by 50-60%on day 14 of treatment.

Methods of treating a patient diagnosed with sickle cell disease, andPKR Activating Compounds for use in such methods, can also includeadministering to the patient the PKR Activating Compound (e.g., acomposition comprising one or more compounds of Formula I, such asCompound 1 or a mixture of Compound 1 and Compound 2) in a daily amountsufficient to increase the patient's ATP blood levels. In someembodiments, the amount is sufficient to increase ATP blood levels by atleast 40% on day 14 of treatment, or greater (e.g., at least 50% on day14 of treatment). In some embodiments, the amount is sufficient toincrease ATP blood levels by 40-65% on day 14 of treatment. In someembodiments, the amount is sufficient to increase ATP blood levels by atleast 50% on day 14 of treatment, or greater (e.g., at least 50% on day14 of treatment). In some embodiments, the amount is sufficient toincrease ATP blood levels by 50-65% on day 14 of treatment.

In some examples, a pharmaceutical composition comprising Compound 1 canbe used in a method of treating a patient diagnosed with sickle celldisease, the method comprising administering to the patient 400 mg ofCompound 1 once per day (QD)

In some examples, a pharmaceutical composition comprising Compound 1 canbe used in a method of treating a patient diagnosed with sickle celldisease, the method comprising administering to the patient 200 mg ofCompound 1 twice per day (BID)

In some embodiments, the present disclosure provides PKR ActivatingCompounds of Formula I:

or a pharmaceutically acceptable salt thereof. In some embodiments, aPKR Activating Compound is1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

The compound of Formula I is preferably Compound 1:

or a pharmaceutically acceptable salt thereof. In some embodiments, acompound of Formula I is(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

In some embodiments, the present disclosure provides a PKR ActivatingCompound that is any of the compounds listed in FIG. 1, or apharmaceutically acceptable salt thereof.

The present disclosure also provides compositions (e.g. pharmaceuticalcompositions) comprising a compound of Formula I. In some embodiments, aprovided composition containing a compound of Formula I comprises amixture of Compound 1 and Compound 2:

or a pharmaceutically acceptable salt thereof. The present disclosurealso provides compositions (e.g. pharmaceutical compositions) comprisingany of the compounds listed in FIG. 1, or a pharmaceutically acceptablesalt thereof.

Pharmaceutical compositions comprising a PKR Activating Compositioncontaining a compound of Formula (I) can be formulated for oraladministration (e.g., as a capsule or tablet). For example, Compound 1can be combined with suitable compendial excipients to form an oral unitdosage form, such as a capsule or tablet, containing a target dose ofCompound 1. The drug product can be prepared by first manufacturingCompound 1 as an active pharmaceutical ingredient (API), followed byroller compaction/milling with intragranular excipients and blendingwith extra granular excipients. A Drug Product can contain the Compound1 API and excipient components in Table 1 in a tablet in a desireddosage strength of Compound 1 (e.g., a 25 mg or 100 mg tablet formedfrom a Pharmaceutical Composition in Table 1). The blended material canbe compressed to form tablets and then film coated.

The pharmaceutical composition preferably comprises about 30-70% byweight of(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,and a pharmaceutically acceptable excipient in an oral dosage form.

TABLE 1 Exemplary Pharmaceutical Compositions of Compound 1 for OralAdministration % Formulation Function (weight) Examplary Component API  30-70% Compound 1 Filler   15-40% Microcrystalline Cellulose Drybinder  2-10% Crospovidone Kollidon CL Glidant 0.25-1.25% Colloidal SiliconDioxide Lubricant 0.25-1.00% Magnesium Stearate, Hyqual

In some embodiments, a provided composition containing a compound ofFormula I comprises a mixture of(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-oneand(R)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.In some embodiments, a provided composition containing a compound ofFormula I is a mixture of Compound 1 and Compound 2 as part of a PKRActivating Composition. In some embodiments, a compound of Formula I isracemic. In some embodiments, a compound of Formula I consists of about50% of Compound 1 and about 50% of Compound 2. In some embodiments, acompound of Formula I is not racemic. In some embodiments, a compound ofFormula I does not consist of about 50% of Compound 1 and about 50% ofCompound 2. In some embodiments, a compound of Formula I comprises about99-95%, about 95-90%, about 90-80%, about 80-70%, or about 70-60% ofCompound 1. In some embodiments, a compound of Formula I comprises about99%, 98%, 95%, 90%, 80%, 70%, or 60% of Compound 1.

In some embodiments, a PKR Activating Composition comprises a mixture ofCompound 1 and Compound 2. In some embodiments, a PKR ActivatingComposition comprises a mixture of Compound 1 and Compound 2, whereinthe PKR Activating Composition comprises a therapeutically effectiveamount of Compound 1.

Compositions comprising a compound of Formula I can be prepared as shownin FIG. 4A and FIG. 4B. Compounds of Formula I can be obtained by thegeneral chemical synthesis scheme of FIG. 4A. Compound 1 can be obtainedby the chemical synthesis route of FIG. 4A or FIG. 4B. In brief,compounds of Formula I (FIG. 4A) and/or Compound 1 (FIG. 4B) can beobtained from a series of four reaction steps from commerciallyavailable starting materials. Commercially available7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine was treated with a mixture ofn-butyl lithium and dibutylmagnesium followed by sulfuryl chloride togive sulfonyl chloride 3. Treatment of 3 with tert-butyl1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate in the presence oftriethylamine (TEA) afforded Boc-protected monosulfonamide 4. Compound 4was then de-protected in the presence of trifluoroacetic acid (TFA) togive 5, the free base of the monosulfonamide. The last step to generateCompound 1 (FIG. 4B) or Compound 1 and Compound 2 (FIG. 4A) was an amidecoupling of 5 and tropic acid in the presence of1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium3-oxide hexafluoro-phosphate (HATU).

The compounds listed in FIG. 1 can be prepared as shown in FIG. 4C andas described in International Publication No. WO 2018/175474, publishedSep. 27, 2018. Generally, the compounds listed in FIG. 1 can be preparedby acylation and sulfonylation of the secondary amine groups ofhexahydropyrrolopyrrole 6. For example, sulfonylation of 6 with asuitable sulfonyl chloride 7 affords sulfonyl hexahydropyrrolopyrrole 8,which is then treated with a suitable carboxylic acid 9 in the presenceof an amide coupling reagent (e.g., HATU) to afford compound 10 (Path1). Alternatively, acylation of 6 with a suitable carboxylic acid 9 inthe presence of an amide coupling reagent affords acylhexahydropyrrolopyrrole 11, which is then treated with a suitablesulfonyl chloride 7 to afford compound 10 (Path 2). As a person ofordinary skill would understand, well-known protecting groups,functionalization reactions, and separation techniques can be used inconjunction with Paths 1 and 2 to obtain the specific compounds listedin FIG. 1.

Methods of treating SCD also include administration of a therapeuticallyeffective amount of a bioactive compound (e.g., a small molecule,nucleic acid, or antibody or other therapy) that reduces HgbSpolymerization, for example by increasing HgbS affinity for oxygen.

In other embodiments, the disclosure relates to each of the followingnumbered embodiments:

1. A composition comprising a PKR Activating Compound of Formula I, or apharmaceutically acceptable salt thereof:

2. The composition of embodiment 1, wherein the compound of Formula I isCompound 1, or a pharmaceutically acceptable salt thereof:

3. The composition of embodiment 2, wherein the composition comprises amixture of Compound 1 and Compound 2, or a pharmaceutically acceptablesalt thereof:

4. The composition of embodiment 1, comprising the compound:1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

5. The composition of any one of embodiments 1-4, formulated as an oralunit dosage form.

6. A method of treating a patient diagnosed with a sickle cell disease(SCD), the method comprising administering to the patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising(S)-1-(5-((2,3-dihydro[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,or a pharmaceutically acceptable salt thereof.

7. The method of embodiment 6, wherein the method comprises oraladministration of the pharmaceutical composition comprising(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,as the only PKR Activating Compound in the pharmaceutical composition.

8. A method of treating a patient diagnosed with a sickle cell disease(SCD), the method comprising administering to the patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising Compound 1:

or a pharmaceutically acceptable salt thereof.

9. A composition comprising a compound of Formula I obtainable by aprocess comprising the step of converting compound 5 into a compound ofFormula I in a reaction described as Step 4:

10. The composition of embodiment 9, wherein the process furthercomprises first obtaining the compound 5 from a compound 4 by a processcomprising Step 3:

11. The composition of embodiment 10, wherein the process furthercomprises first obtaining the compound 4 from a compound 3 by a processcomprising Step 2:

12. The composition of embodiment 11, wherein the process furthercomprises first obtaining the compound 3 from a process comprising Step1:

13. A method of treating a patient diagnosed with sickle cell disease(SCD), the method comprising administering to the patient in needthereof a therapeutically effective amount of a PKR Activating Compoundhaving an AC₅₀ value of less than 1 μM using the Luminescence Assaydescribed in Example 2.

14. The method of embodiment 13, wherein the PKR Activating Compound isCompound 1.

15. The method of any one of embodiments 13-14, wherein the PKRActivating Compound is orally administered to the patient in needthereof.

16. The use of Compound 1:

or a pharmaceutically acceptable salt thereof, for the treatment ofpatients diagnosed with sickle cell disease (SCD).

17. The use of a PKR Activating Compound having an AC₅₀ value of lessthan 1 μM using the Luminescence Assay described in Example 2, in thetreatment of patients diagnosed with sickle cell disease.

18. The method of any one of embodiments 6-8 or 13-15, comprising theadministration of Compound 1 once per day.

19. The method of any one of embodiments 6-8 or 13-15, comprising theadministration of a total of 25 mg-1,500 mg of Compound 1 each day.

20. The method of any one of embodiments 18-19, comprising theadministration of a total of 25 mg-130 mg of Compound 1 each day.

In other embodiments, the disclosure relates to each of the followingnumbered embodiments:

1. A method for reducing 2,3-diphosphoglycerate (2,3-DPG) levels in apatient's red blood cells, comprising administering to the patient a PKRActivating Compound in a therapeutically effective amount, wherein thePKR Activating Compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, having an AC₅₀ value ofless than 1 μM using the Luminescence Assay described in Example 2.

2. The method of embodiment 1, wherein the PKR Activating Compound isCompound 1:

or a pharmaceutically acceptable salt thereof.

3. The method of embodiment 1, wherein the PKR Activating Compound isCompound 1:

4. The method of embodiment 3, wherein the PKR Activating Compound isadministered in an amount of 25-1500 mg per day.

5. The method of embodiment 3, wherein the PKR Activating Compound isadministered once daily in an amount of 250 mg, 300 mg, 500 mg, 600 mg,1000 mg, or 1500 mg per day.

6. The method of embodiment 3, wherein the PKR Activating Compound isadministered once daily in an amount of 100 mg per day.

7. The method of embodiment 3, wherein the PKR Activating Compound isadministered once daily in an amount of 600 mg per day.

8. The method of embodiment 3, wherein the PKR Activating Compound isadministered once per day.

9. The method of embodiment 3, wherein the PKR Activating Compound isorally administered to the patient.

10. The method of embodiment 3, wherein Compound 1 is the only PKRActivating Compound administered to the patient.

In other embodiments, the disclosure relates to each of the followingnumbered embodiments:

1. A method for reducing 2,3-diphosphoglycerate (2,3-DPG) levels in apatient's red blood cells, comprising administering to the patient thePKR Activating Compound in an amount sufficient to reduce 2,3-DPG levelsin the patient's red blood cells by at least 30% after 24 hours, whereinthe PKR Activating Compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, having an AC₅₀ value ofless than 1 μM using the Luminescence Assay described in Example 2.

2. The method of embodiment 1, wherein the PKR Activating Compound isCompound 1:

or a pharmaceutically acceptable salt thereof.

3. The method of embodiment 1, wherein the PKR Activating Compound isCompound 1:

4. The method of embodiment 1, wherein Compound 1 is the only PKRActivating Compound administered to the patient.

5. The method of any one of embodiments 1-4, wherein the PKR ActivatingCompound is orally administered to the patient.

6. The method of any one of embodiments 1-5, wherein the PKR ActivatingCompound is administered once per day.

7. The method of any one of embodiments 1-6, wherein the PKR ActivatingCompound is administered in an amount sufficient to reduce 2,3-DPGlevels in the patient's red blood cells by at least 40% after 24 hours.

8. The method of any one of embodiments 1-7, wherein the PKR ActivatingCompound is administered in a daily amount sufficient to increase thepatient's ATP blood levels by at least 40% on day 14 of treatment.

9. The method of any one of embodiments 1-5, wherein the PKR ActivatingCompound is administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg,700 mg, 1100 mg, or 1500 mg per day.

10. The method of any one of embodiments 1-5, wherein the PKR ActivatingCompound is administered in an amount of 200 mg per day.

11. The method of embodiment 10, wherein the PKR Activating Compound isadministered in an amount of 200 mg per day once per day (QD).

12. The method of embodiment 10, wherein the PKR Activating Compound isadministered in an amount of 100 mg per day twice per day (BID).

13. The method of any one of embodiments 1-5, wherein the PKR ActivatingCompound is administered in an amount of 400 mg per day.

14. The method of embodiment 13, wherein the PKR Activating Compound isadministered in an amount of 400 mg once per day (QD).

15. The method of embodiment 13, wherein the PKR Activating Compound isadministered in an amount of 200 mg twice per day (BID).

16. The method of any one of embodiments 1-5, wherein the PKR ActivatingCompound is administered in an amount of 600 mg per day.

17. The method of embodiment 16, wherein the PKR Activating Compound isadministered in an amount of 300 mg twice per day (BID).

18. The method of any one of embodiments 1-5, wherein the PKR ActivatingCompound is administered in an amount of 700 mg per day.

19. The method of embodiment 18, wherein the PKR Activating Compound isadministered in an amount of 700 mg once per day (QD).

30. The method of embodiment 18, wherein the PKR Activating Compound isadministered in an amount of 350 mg twice per day (BID).

The present disclosure enables one of skill in the relevant art to makeand use the inventions provided herein in accordance with multiple andvaried embodiments. Various alterations, modifications, and improvementsof the present disclosure that readily occur to those skilled in theart, including certain alterations, modifications, substitutions, andimprovements are also part of this disclosure. Accordingly, theforegoing description and drawings are by way of example to illustratethe discoveries provided herein.

EXAMPLES

As the enzyme that catalyzes the last step of glycolysis, PKR underliesreactions that directly impact the metabolic health and primaryfunctions of RBCs. The following Examples demonstrate how PKR activationby Compound 1 impacts RBCs. The primary effect of Compound 1 on RBCs isa decrease in 2,3-DPG that is proposed to reduce Hgb sickling and itsconsequences on RBCs and oxygen delivery to tissues. Compound 1 alsoincreases ATP, which may provide metabolic resources to support cellmembrane integrity and protect against loss of deformability andincreased levels of hemolysis in SCD. With the combination of effectsCompound 1 has on RBCs, it is likely to reduce the clinical sequelae ofsickle Hgb and provide therapeutic benefits for patients with SCD.

The PKR Activating Compound designated Compound 1 was prepared asdescribed in Example 1, and tested for PKR activating activity in thebiochemical assay of Example 2.

The biological enzymatic activity of PKR (i.e., formation of ATP and/orpyruvate) was evaluated in enzyme and cell assays with Compound 1, asdescribed in Example 3 and Example 4, respectively. Results from enzymeassays show that Compound 1 is an activator of recombinant wt-PKR andmutant PKR, (e.g., R510Q), which is one of the most prevalent PKRmutations in North America. PKR exists in both a dimeric and tetramericstate, but functions most efficiently as a tetramer. Compound 1 is anallosteric activator of PKR and is shown to stabilize the tetramericform of PKR, thereby lowering the K_(m) (the Michaelis-Menten constant)for PEP.

In vivo testing in mice (Examples 5) demonstrated PKR activation in wtmice, and provided an evaluation of effects on RBCs and Hgb in a murinemodel of SCD. Compound 1 was well tolerated up to the highest dosetested, and exposures increased in a dose-proportional manner. Levels of2,3-DPG were reduced by >30% for doses ≥120 mg/kg Compound 1 (AUC from 0to 24 hours (AUC₀₋₂₄>5200 hr·ng/mL) and levels of ATP were increasedby >40% for ≥60 mg/kg Compound 1 (AUC₀₋₂₄>4000 hr·ng/mL).

In some embodiments, a daily dose of between 100 mg to 1500 mg of a PKRActivating Compound is administered to humans. In some embodiments, adaily dose of between 100 mg to 1500 mg of Compound 1 is administered tohumans. In some embodiments, a daily dose of between 100 mg to 1500 mgof any of the compounds listed in FIG. 1 is administered to humans. Inparticular, a total daily dose of 100 mg-600 mg of a PKR ActivatingCompound can be administered to humans (including, e.g., a dose of 100mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg, per day, in single ordivided doses). In particular, a total daily dose of 100 mg-600 mg ofCompound 1 can be administered to humans (including, e.g., a dose of 100mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg, per day, in single ordivided doses). In particular, a total daily dose of 100 mg-600 mg ofany of the compounds listed in FIG. 1 can be administered to humans(including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or600 mg, per day, in single or divided doses). In some embodiments, adaily dose of 400 mg (e.g., 400 mg QD or 200 mg BID) of a PKR ActivatingCompound is administered to humans. In some embodiments, a daily dose of400 mg (e.g., 400 mg QD or 200 mg BID) of Compound 1, or apharmaceutically acceptable salt thereof, is administered to humans. Insome embodiments, a daily dose of 400 mg (e.g., 400 mg QD or 200 mg BID)of any of the compounds listed in FIG. 1 is administered to humans.

Example 1: Synthesis of Compounds of Formula I

The PKR Activating Compound 1 was obtained by the method describedherein and the reaction scheme shown in FIG. 4A and/or FIG. 4B. Compound1 has a molecular weight of 457.50 Da.

Step 1. 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (3)

Into a 100 mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed a solution of n-BuLi in hexane (2.5 M,2 mL, 5.0 mmol, 0.54 equiv) and a solution of n-Bu₂Mg in heptanes (1.0M, 4.8 mL, 4.8 mmol, 0.53 equiv). The resulting solution was stirred for10 min at RT (20° C.). This was followed by the dropwise addition of asolution of 7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine (2 g, 9.26 mmol,1.00 equiv) in tetrahydrofuran (16 mL) with stirring at −10° C. in 10min. The resulting mixture was stirred for 1 h at −10° C. The reactionmixture was slowly added to a solution of sulfuryl chloride (16 mL) at−10° C. The resulting mixture was stirred for 0.5 h at −10° C. Thereaction was then quenched by the careful addition of 30 mL of saturatedammonium chloride solution at 0° C. The resulting mixture was extractedwith 3×50 mL of dichloromethane. The organic layers were combined, driedover anhydrous sodium sulfate, filtered and concentrated under vacuum.The residue was purified by silica gel column chromatography, elutingwith ethyl acetate/petroleum ether (1:3). This provided 1.3 g (60%) of2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride as a white solid.LCMS m/z: calculated for C₇H₆ClNO₄S: 235.64; found: 236 [M+H]⁺.

Step 2. tert-Butyl5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate(4)

Into a 100-mL round-bottom flask was placed2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (1.3 g, 5.52 mmol,1.00 equiv), tert-butyl1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate (1.16 g, 5.52mmol), dichloromethane (40 mL), and triethylamine (1.39 g, 13.74 mmol,2.49 equiv). The solution was stirred for 2 h at 20° C., then dilutedwith 40 mL of water. The resulting mixture was extracted with 3×30 mL ofdichloromethane. The organic layers were combined, dried over anhydroussodium sulfate, filtered and concentrated under vacuum. The residue waspurified by silica gel column chromatography, eluting withdichloromethane/methanol (10:1). This provided 1.2 g (53%) of tert-butyl5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylateas a yellow solid. LCMS m/z: calculated for C₁₈H₂₃N₃O₆S: 409.46; found:410 [M+H]⁺.

Step 3.2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole(5)

Into a 100-mL round-bottom flask was placed tert-butyl5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate(1.2 g, 2.93 mmol, 1.00 equiv), dichloromethane (30 mL), andtrifluoroacetic acid (6 mL). The solution was stirred for 1 h at 20° C.The resulting mixture was concentrated under vacuum. The residue wasdissolved in 10 mL of methanol and the pH was adjusted to 8 with sodiumbicarbonate (2 mol/L). The resulting solution was extracted with 3×10 mLof dichloromethane. The organic layers were combined, dried overanhydrous sodium sulfate, filtered and concentrated under vacuum. Thecrude product was purified by silica gel column chromatography, elutingwith dichloromethane/methanol (10:1). This provided 650 mg (72%) of2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrroleas a yellow solid. LCMS m/z: calculated for C₁₃H₁₅N₃O₄S: 309.34; found:310 [M+H]⁺.

Step 4.(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(1) and(R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(2)

Into a 100 mL round-bottom flask was placed2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole(150 mg, 0.48 mmol, 1.00 equiv), 3-hydroxy-2-phenylpropanoic acid (97mg, 0.58 mmol, 1.20 equiv), dichloromethane (10 mL), HATU (369 mg, 0.97mmol, 2.00 equiv) and DIEA (188 mg, 1.46 mmol, 3.00 equiv). Theresulting solution was stirred overnight at 20° C. The reaction mixturewas diluted with 20 mL of water and was then extracted with 3×20 mL ofdichloromethane. The organic layers were combined, dried over anhydroussodium sulfate, filtered and concentrated under vacuum. The residue waspurified by prep-TLC eluted with dichloromethane/methanol (20:1) andfurther purified by prep-HPLC (Column: XBridge C18 OBD Prep Column, 100Å, 5 μm, 19 mm×250 mm; Mobile Phase A: water (10 mmol/L NH₄HCO₃), MobilePhase B: MeCN; Gradient: 15% B to 45% B over 8 min; Flow rate: 20mL/min; UV Detector: 254 nm). The two enantiomers were separated byprep-Chiral HPLC (Column, Daicel CHIRALPAK® IF, 2.0 cm×25 cm, 5 μm;mobile phase A: DCM, phase B: MeOH (hold 60% MeOH over 15 min); Flowrate: 16 mL/min; Detector, UV 254 & 220 nm). This resulted in peak 1 (2,Rt: 8.47 min) 9.0 mg (4%) of(R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-oneas a yellow solid; and peak 2 (1, Rt: 11.83 min) 10.6 mg (5%) of(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-oneas a yellow solid.

(1): ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J=2.0 Hz, 1H), 7.61 (d, J=2.0Hz, 1H), 7.31-7.20 (m, 5H), 4.75 (t, J=5.2 Hz, 1H), 4.50-4.47 (m, 2H),4.40-4.36 (m, 1H), 4.32-4.29 (m, 2H), 4.11-3.87 (m, 8H), 3.80-3.77 (m,1H), 3.44-3.41 (m, 1H). LC-MS (ESI) m/z: calculated for C₂₂H₂₃N₃O₆S:457.13; found: 458.0 [M+H]⁺.

(2): ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J=2.0 Hz, 1H), 7.60 (d, J=2.0Hz, 1H), 7.31-7.18 (m, 5H), 4.75 (t, J=5.2 Hz, 1H), 4.52-4.45 (m, 2H),4.40-4.36 (m, 1H), 4.34-4.26 (m, 2H), 4.11-3.87 (m, 8H), 3.80-3.78 (m,1H), 3.44-3.43 (m, 1H). LC-MS (ESI) m/z: calculated for C₂₂H₂₃N₃O₆S:457.13; found: 458.0 [M+H]⁺.

Step 5.(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(1)

Alternatively, Compound 1 can be synthesized using the proceduredescribed here as Step 5. A solution of7-((3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine(130.9 mg, 0.423 mmol) in DMF (2.5 ml) was cooled on an ice bath, thentreated with (S)-3-hydroxy-2-phenylpropanoic acid (84.8 mg, 0.510 mmol),HATU (195.5 mg, 0.514 mmol), and DIEA (0.30 mL, 1.718 mmol) and stirredat ambient temperature overnight. The solution was diluted with EtOAc(20 mL), washed sequentially with water (20 mL) and brine (2×20 mL),dried (MgSO₄), filtered, treated with silica gel, and evaporated underreduced pressure. The material was chromatographed by Biotage MPLC (10 gsilica gel column, 0 to 5% MeOH in DCM) to provide a white, slightlysticky solid. The sample was readsorbed onto silica gel andchromatographed (10 g silica gel column, 0 to 100% EtOAc in hexanes) toprovide(2S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one(106.5 mg, 0.233 mmol, 55% yield) as a white solid.

Example 2: Biochemical Assay for Identification of PKR ActivatingActivity

PKR Activating Compounds can be identified with the biochemicalLuminescence Assay of Example 2. The PKR activating activity of a seriesof chemical compounds was evaluated using the Luminescence Assay below,including compounds designated Compound 1, Compound 2, and Compounds 6,7, and 8 below, and the compounds listed in FIG. 1.

For each tested compound, the ability to activate PKR was determinedusing the following Luminescence Assay. The effect of phosphorylation ofadenosine-5′-diphosphate (ADP) by PKR is determined by the Kinase GloPlus Assay (Promega) in the presence or absence of FBP(D-fructose-1,6-diphosphate; BOC Sciences, CAS: 81028-91-3) as follows.Unless otherwise indicated, all reagents are purchased fromSigma-Aldrich. All reagents are prepared in buffer containing 50 mMTris-HCl, 100 mM KCl, 5 mM MgCl₂, and 0.01% Triton X100, 0.03% BSA, and1 mM DTT. Enzyme and PEP (phosphoenolpyruvate) are added at 2× to allwells of an assay-ready plate containing serial dilutions of testcompounds or DMSO vehicle. Final enzyme concentrations for PKR(wt),PKR(R510Q), and PKR(G332S) are 0.8 nM, 0.8 nM, and 10 nM respectively.Final PEP concentration is 100 μM. The Enzyme/PEP mixture is incubatedwith compounds for 30 minutes at RT before the assay is initiated withthe addition of 2×ADP and KinaseGloPlus. Final concentration of ADP is100 μM. Final concentration of KinaseGloPlus is 12.5%. For assayscontaining FBP, that reagent is added at 30 μM upon reaction initiation.Reactions are allowed to progress for 45 minutes at RT untilluminescence is recorded by the BMG PHERAstar FS Multilabel Reader. Thecompound is tested in triplicate at concentrations ranging from 42.5 μMto 2.2 nM in 0.83% DMSO. AC₅₀ measurements were obtained by the standardfour parameter fit algorithm of ActivityBase XE Runner (max, min, slopeand AC₅₀). The AC₅₀ value for a compound is the concentration (μM) atwhich the activity along the four parameter logistic curve fit ishalfway between minimum and maximum activity.

As set forth in Tables 2 and 3 below and in FIG. 1, AC₅₀ values aredefined as follows: ≤0.1 μM (+++); >0.1 μM and ≤1.0 μM (++); >1.0 μM and≤40 μM (+); >40 μM (0).

TABLE 2 Luminescence Assay Data AC₅₀ AC₅₀ AC₅₀ Compound (PKRG332S)(PKRR510Q) (WT) 1 ++ +++ +++ 2 + + +

TABLE 3 Additional Luminescence Assay Data AC₅₀ AC₅₀ Compound Structure(PKRG332S) (PKRR510Q) 6

++ + 7

0 0 8

0 0

Compounds and compositions described herein are activators of wild typePKR and certain PKR mutants having lower activities compared to the wildtype. Such mutations in PKR can affect enzyme activity (catalyticefficiency), regulatory properties, and/or thermostability of theenzyme. One example of a PKR mutation is G332S. Another example of a PKRmutation is R510Q.

Example 3: Enzyme Assays of a PKR Activating Compound

The effect of 2 μM Compound 1 on maximum velocity (V_(max)) and PEPK_(m) (Michaelis-Menten constant, i.e., the concentration of PEP atwhich v=½v_(max)) was evaluated for wt-PKR and PKR-R510Q. Tests wereconducted in the presence and absence of fructose-1,6-bisphosphate(FBP), a known allosteric activator of PKR. Assessments were made up to60 min at RT, and V_(max) and PEP K_(m) were calculated. The effect ofCompound 1 on V_(max) ranged from no effect to a modest increase (seeFIG. 5 for a representative curve). Compound 1 consistently reduced thePEP K_(m), typically by ˜2 fold, for wt-PKR and PKR-R510Q in thepresence or absence of FBP (Table 4), demonstrating that Compound 1 canenhance the rate of PKR at physiological concentrations of PEP.

TABLE 4 Effect of Compound 1 on PKR Enzyme Kinetic Parameters No FBP 30μM FBP Kinetic 2 μM 2 μM Enzyme Parameter^(a) DMSO Compound 1 DMSOCompound 1 WT- V_(max) 1.00 1.14 1.19 1.16 PKR PEP K_(m) 4.84 2.44 1.981.00 PKR V_(max) 1.54 1.56 1.00 1.29 R510Q PEP K_(m) 6.20 1.70 2.01 1.00^(a)All values in Table 4 are normalized to 1.00, relative to the othervalues in the same row

Activation of wt-PKR and PKR-R510Q by different concentrations ofCompound 1 was evaluated for PEP concentrations at or below K_(m).Compound 1 increased the rate of ATP formation, with AC₅₀ values rangingfrom <0.05 to <0.10 μM and a range of <2.0 to <3.0 maximum-foldactivation (i.e., <200% to <300%) (Table 5). Representative data fromPKR-R510Q showed that the effect was concentration dependent (FIG. 6).

TABLE 5 Activation of PKR Wild and Mutant Types by Compound 1Maximum-fold PK Enzyme Activation AC₅₀ (μM) WT-PKR <2.0 <0.05 PKR R510Q<3.0 <0.10

Example 4: Cell Assays of a PKR Activating Compound

The activation of wt-PKR by Compound 1 in mature human erythrocytes exvivo was evaluated in purified RBCs purchased from Research BloodComponents. Cells treated with Compound 1 for 3 hr in glucose-containingmedia were washed, lysed, and assayed using a Biovision Pyruvate KinaseAssay (K709-100). The assay was repeated multiple times to account fordonor-to-donor variability and the relatively narrow dynamic range. Meanmaximum activation increase (Max-Min) was <100% and mean 50% effectiveconcentration (EC₅₀) was <125 nM (Table 6). wt-PKR was activated in aconcentration-dependent manner (FIG. 7).

TABLE 6 Wild Type PKR Activation in Human Red Blood Cells Treated withCompound 1 Replicate Max-Min (%) EC₅₀ (nM) 1 <125 <250 2 <150 <150 3<100 <50 4 <50 <50 Mean <100 <125

Mouse RBCs were isolated fresh from whole blood using a Ficoll gradientand assayed with methods similar to those used in the human RBCs assays.Maximum activation increase, and EC₅₀ values were comparable to theeffects in human RBCs (Table 7).

TABLE 7 Effect of Compound 1 on PKR Activation in Mouse Red Blood CellsReplicate Max-Min (%) EC₅₀ (nM) 1 <50 <125 2 <100 <125 Mean <100 <125

Example 5: Pharmacokinetic/Pharmacodynamic Studies of Compound 1 in WildType Mice

Two pharmacokinetic (PK)/phamacodynamic (PD) studies were conducted inBalb/c mice that were administered Compound 1 once daily by oral gavage(formulated in 10% Cremophor EL/10% PG/80% DI water) for 7 days (QD×7)at doses of 0 (vehicle), 3.75, 7.5, 15, 30, 60 mg/kg (Study 1); 0(vehicle), 7.5, 15, 30, 60, 120, or 240 mg/kg (Study 2). On the 7th day,whole blood was collected 24 hours after dosing and snap frozen. Sampleswere later thawed and analyzed by LC/MS for 2,3-DPG and ATP levels. Inboth studies, Compound 1 was well tolerated. No adverse clinical signswere observed and there were no differences in body weight changecompared with the vehicle group.

The levels of 2,3-DPG decreased with Compound 1 treatment (FIGS. 8A and8B (Studies 1 and 2) and FIG. 9 (Study 2)). In general, reductionswere >20% at ≥15 mg/kg Compound 1, and >30% for 120 and 240 mg/kgCompound 1. Together, the results from the highest doses provide in vivoevidence that 2,3-DPG decreases with PKR activation.

Evaluation of ATP levels in these studies showed that treatment withCompound 1 increased levels of ATP. In Study 1, ATP increased 21% and79% with 30 and 60 mg/kg Compound 1, respectively, compared to vehicle,and in Study 2, ATP levels increased with exposure with doses up to 120mg/kg Compound 1 with a maximum increase of ˜110% compared to vehicle(FIG. 10A and FIG. 10B). At the highest dose, 240 mg/kg Compound 1, ATPlevels increased by 45%. Levels of ATP correlated with Compound 1exposure in a manner similar across both studies.

Example 6: A SAD/MAD Study to Assess the Safety, Pharmacokinetics, andPharmacodynamics of Compound 1 in Healthy Volunteers and Sickle CellDisease Patients

Compound 1 will be evaluated in a randomized, placebo-controlled, doubleblind, single ascending and multiple ascending dose study to assess thesafety, pharmacokinetics, and pharmacodynamics of Compound 1 in healthyvolunteers and sickle cell disease patients. The use of Compound 1 isdisclosed herein for treatment of sickle cell disease in humans.

Compound 1 is an oral small-molecule agonist of pyruvate kinase redblood cell isozyme (PKR) being developed for the treatment of hemolyticanemias. This human clinical trial study will characterize the safety,tolerability and the pharmacokinetics/pharmacodynamics (PK/PD) of asingle ascending dose and multiple ascending doses of Compound 1 in thecontext of phase 1 studies in healthy volunteers and sickle cell diseasepatients. The effects of food on the absorption of Compound 1 will alsobe evaluated, in healthy volunteers.

The objectives of the study include the following:

-   -   1. To evaluate the safety and tolerability of a single ascending        dose and multiple ascending doses of Compound 1 in healthy        volunteers and sickle cell disease (SCD) patients.    -   2. To characterize the pharmacokinetics (PK) of Compound 1.    -   3. To evaluate the levels of 2,3-diphosphoglycerate (DPG) and        adenosine triphosphate (ATP) in the red blood cells (RBCs) of        healthy volunteers and SCD patients after single and multiple        doses of Compound 1.    -   4. To evaluate the relationship between Compound 1 plasma        concentration and potential effects on the QT interval in        healthy volunteers.    -   5. To evaluate the effect of single ascending doses of Compound        1 on other electrocardiogram (ECG) parameters (heart rate, PR        and QRS interval and T-wave morphology) in healthy volunteers.    -   6. To explore food effects on the PK of Compound 1 in healthy        volunteers.    -   7. To explore the association of Compound 1 exposure and        response variables (such as safety, pharmacodynamics (PD),        hematologic parameters as appropriate).    -   8. To explore effects of Compound 1 after single and multiple        doses on RBC function.    -   9. To explore effects of Compound 1 after multiple doses in SCD        patients on RBC metabolism, inflammation and coagulation.

This is a first-in-human (FIH), Phase 1 study of Compound 1 that willcharacterize the safety, PK, and PD of Compound 1 after a single doseand after repeated dosing first in healthy adult volunteers and then inadolescents or adults with sickle cell disease. The study arms andassigned interventions to be employed in the study are summarized inTable 8. Initially, a dose range of Compound 1 in single ascending dose(SAD) escalation cohorts will be explored in healthy subjects.Enrollment of healthy subjects into 2-week multiple ascending dose (MAD)escalation cohorts will be initiated once the safety and PK from atleast two SAD cohorts is available to inform the doses for the 2-weekMAD portion of the study. The MAD cohorts will then run in parallel tothe single dose cohorts. A single dose cohort is planned to understandfood effects (FE) on the PK of Compound 1. After the SAD and FE studiesin healthy subjects are completed, the safety, PK and PD of a singledose of Compound 1 that was found to be safe in healthy subjects willthen be evaluated in sickle cell disease (SCD) subjects. Multiple dosestudies in SCD subjects will then be initiated upon completion of MADstudies in healthy volunteers. Compound 1 will be administered in 25 mgand 100 mg tablets delivered orally.

TABLE 8 Arms Assigned Interventions Experimental: Single ascending doseDrug: Compound 1/Placebo cohorts in healthy subjects Healthy volunteersubjects Healthy volunteer subject cohorts will receive Compound 1/randomized 6:2 receiving a single dose of placebo and be monitoredCompound 1 or placebo. The first cohort for side effects while willreceive 200 mg of Compound 1 or undergoing pharmacokinetic placebo. Doseescalation will occur if and pharmacodynamics Compound 1 or placebo istolerated. The studies maximum dose of Compound 1 or placebo will be1500 mg. Planned doses for the SAD cohorts are listed in Table 9.Experimental: Multiple ascending dose Drug: Compound 1/ cohorts inhealthy subjects Placebo Healthy volunteer subject cohorts Healthyvolunteer randomized 9:3 to receive Compound 1 or subjects will receiveplacebo for 14 days continuous dosing. Compound 1/placebo and The firstcohort will receive 100 mg of be monitored for side Compound 1 orplacebo daily X 14 days. effects while undergoing Alternatively, thefirst cohort will receive pharmacokinetics and 200 mg (e.g., 100 mg BIDor 200 mg QD) pharmacodynamic studies of Compound 1 or placebo daily X14 days. The maximum dose of Compound 1/placebo will be 600 mg Compound1/placebo daily for 14 days. Planned doses for the MAD cohorts arelisted in Table 10. Experimental: Food Effect Cohort in Drug: Compound 1healthy subjects Healthy subjects will Healthy Volunteer subject cohortof 10 receive Compound 1 with subjects who will receive a single dose ofor without food and Compound 1 with food and without food. undergopharmacokinetic Dose will be administered per the protocol studiesdefined dose. Healthy Volunteer subject cohort of 10 subjects who willreceive a single dose of Compound 1 with food and without food. Dosewill be 500 mg of Compound 1, but is subject to change based on thepharmacokinetic profile of Compound 1 observed in the initial SADcohorts and the safety profile of Compound 1 observed in prior SAD andMAD cohorts. Experimental: Single ascending dose Drug: Compound 1/cohorts in SCD subjects Placebo Sickle cell disease subject cohort SCDsubjects randomized 6:2 receiving a single dose of will receive CompoundCompound 1 or placebo. The dose of 1/placebo and be Compound 1/placeboadministered will be monitored for side a dose that was found to be safein healthy effects while undergoing subjects. The dose of Compoundpharmacokinetic and 1/placebo administered also will be a dosepharmacodynamics that was found to be studies pharmacodynamically active(e.g., results in a reduction in 2,3-DPG) in healthy subjects.Experimental: Multiple ascending dose Drug: Compound 1/ cohorts in SCDsubjects Placebo Sickle cell disease subject cohorts SCD subjectsrandomized 9:3 to receive Compound 1 or will receive Compound placebofor 14 days continuous dosing. 1/placebo and be The dose of Compound1/placebo monitored for side administered will be a dose less thaneffects while undergoing maximum tolerable dose evaluated inpharmacokinetic and MAD healthy volunteers. The dose of pharmacodynamicsCompound 1/placebo also will be a dose studies that was found to bepharmacodynamically active (e.g., results in a reduction in RBC 2,3-DPGand increase in RBC ATP) in MAD healthy volunteers.

TABLE 9 Dose Level/Cohort Dose Tablet Strength (#/day) SAD 1  200 mg 100mg (2/day) SAD 2  400 mg 100 mg (4/day) SAD 3  700 mg 100 mg (7/day) SAD4 1100 mg  100 mg (11/day) SAD 5 1500 mg  100 mg (15/day)

TABLE 10 Dose Level/Cohort Dose Tablet Strength (#/day) MAD 1 100 mg 100mg (1/day) or  25 mg (4/day) MAD 2 200 mg 100 mg (2/day) MAD 3 400 mg100 mg (4/day) MAD 4 600 mg 100 mg (6/day)

Outcome Measures Primary Outcome Measures

1. Incidence, frequency, and severity of adverse events (AEs) per CTCAEv5.0 of a single ascending dose and multiple ascending doses of Compound1 in adult healthy volunteers and SCD patients.

-   -   [Time Frame: Up to 3 weeks of monitoring]

2. Maximum observed plasma concentration (Cmax)

-   -   [Time Frame: Up to 3 weeks of testing]

3. Time to maximum observed plasma concentration (Tmax)

-   -   [Time Frame: Up to 3 weeks of testing]

4. Area under the plasma concentration-time curve from time zero untilthe 24-hour time point (AUC0-24)

-   -   [Time Frame: Up to 3 weeks of testing]

5. Area under the plasma concentration-time curve from time zero untillast quantifiable time point (AUC0-last)

-   -   [Time Frame: Up to 3 weeks of testing]

6. Area under the plasma concentration-time curve from time zero toinfinity (AUC0-inf)

-   -   [Time Frame: Up to 3 weeks of testing]

7. Terminal elimination half-life (t½)

-   -   [Time Frame: Up to 3 weeks of testing]

8. Apparent clearance (CL/F)

-   -   [Time Frame: Up to 3 weeks of testing]

9. Apparent volume of distribution (Vd/F)

-   -   [Time Frame: Up to 3 weeks of testing]

10. Terminal disposition rate constant (Lz)

-   -   [Time Frame: Up to 3 weeks of testing]

11. Renal clearance (CIR)

-   -   [Time Frame: Up to 3 weeks of testing]

Secondary Outcome Measures

12. Change from baseline in the levels of 2,3-diphosphoglycerate (DPG)and adenosine triphosphate (ATP) in the red blood cells (RBCs) ofhealthy volunteers and SCD patients after single and multiple doses ofCompound 1.

-   -   [Time Frame: Up to 3 weeks of testing]

13. Model-based estimate of change from baseline QT interval correctedusing Fridericia's correction formula (QTcF) and 90% confidence intervalat the estimated Cmax after a single dose of Compound 1 in healthyvolunteers.

-   -   [Time Frame: up to 7 days]

14. Change from baseline heart rate after a single dose of Compound 1 inhealthy volunteers

-   -   [Time Frame: up to 7 days]

15. Change from baseline PR after a single dose of Compound 1 in healthyvolunteers

-   -   [Time Frame: up to 7 days]

16. Change from baseline QRS after a single dose of Compound 1 inhealthy volunteers

-   -   [Time Frame: up to 7 days]

17. Change from baseline T-wave morphology after a single dose ofCompound 1 in healthy volunteers

-   -   [Time Frame: up to 7 days]

Exploratory Outcome Measures

18. Effect of food on C_(max), AUC₀₋₂₄/AUC_(last)

19. Effect of AUC_(last)/AUC₀₋₂₄, C_(max), minimum plasma concentration(C_(min)), peak-to trough ratio, dose linearity, accumulation ratio onsafety, PD, and hematologic parameters of interest, as assessed byexposure-response analyses

20. Effect of chronic Compound 1 dosing on SCD RBC response to oxidativestress in SCD Patients (including evaluation of glutathione, glutathioneperoxidase and superoxide dismutase levels)

21. Effect of chronic Compound 1 dosing on measurable markers ofinflammation in SCD Patients (C-reactive protein, ferritin, interleukin[IL]-1β, IL-6, IL-8, and tumor necrosis factor-α)

22. Effects of chronic Compound 1 dosing on measurable markers ofhypercoagulation in SCD patients (D-dimer, prothrombin 1.2, andthrombin-antithrombin [TAT] complexes)

Eligibility

-   -   Minimum age: 18 Years (healthy volunteers); 12 Years (SCD        subjects)    -   Maximum age: 60 Years    -   Sex: All    -   Gender Based: No    -   Accepts Healthy Volunteers: Yes

Inclusion Criteria

-   -   Healthy volunteer: subjects must be between 18 and 60 years of        age; SCD: subjects must be between 12 and 50 years of age    -   Subjects must have the ability to understand and sign written        informed consent, which must be obtained prior to any        study-related procedures being completed.    -   Subjects must be in general good health, based upon the results        of medical history, a physical examination, vital signs,        laboratory profile, and a 12-lead ECG.    -   Subjects must have a body mass index (BMI) within the range of        18 kg/m2 to 33 kg/m² (inclusive) and a minimum body weight of 50        kg (healthy volunteer subjects) or 40 kg (SCD subjects)    -   For SCD subjects, sickle cell disease previously confirmed by        hemoglobin electrophoresis or genotyping indicating one of the        following hemoglobin genotypes: Hgb SS, Hgb Sβ⁺-thalassemia, Hgb        Sβ⁰-thalassemia, or Hgb SC    -   All males and females of child bearing potential must agree to        use medically accepted contraceptive regimen during study        participation and up to 90 days after.    -   Subjects must be willing to abide by all study requirements and        restrictions.

Exclusion Criteria

-   -   Evidence of clinically significant medical condition or other        condition that might significantly interfere with the        absorption, distribution, metabolism, or excretion of study        drug, or place the subject at an unacceptable risk as a        participant in this study    -   History of clinically significant cardiac diseases including        condition disturbances    -   Abnormal hematologic, renal and liver function studies    -   History of drug or alcohol abuse

Results (Healthy Volunteers)

Four healthy SAD cohorts were evaluated at doses of 200, 400, 700, and1000 mg, and four healthy MAD cohorts received 200 to 600 mg total dailydoses for 14 days at QD or BID dosing (100 mg BID, 200 mg BID, 300 mgBID, and 400 mg QD). In the food effect (FE) cohort, 10 healthy subjectsreceived 200 mg of Compound 1 QD with and without food.

No serious adverse events (SAEs) or AEs leading to withdrawal werereported in the SAD and MAD cohorts of healthy volunteers. In PKassessments, Compound 1 was rapidly absorbed with a median T_(max) of 1hr postdose. Single dose exposure increased in greater thandose-proportional manner at doses ≥700 mg. In multiple-doses deliveredBID or QD, linear PK was observed across all dose levels (100-300 mgBID, 400 mg QD), and exposure remained steady up to day 14, withoutcumulative effect. Compound 1 exposure under fed/fasted conditions wassimilar.

PD activity was demonstrated at all dose levels evaluated in Compound1-treated subjects (Table 11). Table 11 reports the mean maximumpercentage change in 2,3-DPG and ATP across all doses and timepoints inthe SAD and MAD cohorts. As shown in Table 11, a mean decrease in2,3-DPG, and a mean increase in ATP, relative to baseline, was observedin both the SAD and MAD cohorts. Within 24 hr of a single dose ofCompound 1, a decrease in 2,3-DPG was observed. After 14 days ofCompound 1 dosing these PD effects were maintained along with anincrease in ATP over baseline. Accordingly, the mean maximum reductionin the concentration of 2,3-DPG was at least about 40% in patientsreceiving Compound 1 in the SAD study and at least about 50% in patientsreceiving Compound 1 in the MAD study.

TABLE 11 Summary of Mean Maximum Percent Change in Key PD Measures fromBaseline SAD MAD Placebo Compound 1 Placebo Compound 1 PD MarkerStatistics (N = 8) (N = 24) (N = 12 (N = 36) 2,3-DPG Mean −19.5 −46.8−17.0 −56.3 (95% CI) (−25.0, −14.0) (−50.3, −43.2) (−22.9, −11.1)(−58.9, −53.7) P-value <0.0001 <0.0001 ATP Mean 9.2 24.4 7.2 68.5 (95%CI)  (0.5, 18.0) (18.4, 30.3)  (−0.3, 14.7)   (63.6, 73.3) P-value0.0094 <0.0001

In the SAD cohorts, the subjects' blood 2,3-DPG levels were measuredperiodically after dosing by a qualified LC-MS/MS method for thequantitation of 2,3-DPG in blood. Decreased 2,3-DPG blood levels wereobserved 6 hours following a single dose of Compound 1 at all doselevels (earlier timepoints were not collected). Maximum decreases in2,3-DPG levels generally occurred ˜24 hours after the first dose withthe reduction sustained ˜48-72 hr postdose. Table 12 reports the medianpercentage change in 2,3-DPG blood levels, relative to baseline,measured over time in healthy volunteers after a single dose of Compound1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. Accordingly, themedian reduction in the concentration of 2,3-DPG, relative to baseline,was at least about 30% at all dose levels tested 24 hours afteradministration of the single dose.

TABLE 12 Median Percentage Change in 2,3-DPG Levels Time After Dose DosePlacebo 200 mg 400 mg 700 mg 1000 mg 0 0.0 0.0 0.0 0.0 0.0 6 −7.8 −18−23 −29 −20 8 −7.6 −17 −29 −28 −31 12 −4.0 −25 −40 −41 −44 16 −6.0 −33−35 −46 −50 24 −2.0 −31 −39 −49 −48 36 −6.9 −33 −38 −46 −47 48 −15 −29−31 −48 −47 72 −6.9 −18 −30 −33 −21

FIG. 11 is a graph of the blood 2,3-DPG levels measured over time inhealthy volunteers who received a single dose of Compound 1 (200 mg, 400mg, 700 mg, or 1000 mg) or placebo. As shown in FIG. 11, healthyvolunteers who received Compound 1 experienced a decrease in blood2,3-DPG levels, relative subjects who received the placebo. FIG. 12 is agraph of the blood 2,3-DPG levels measured 24 hours post-dose in healthyvolunteers who received a single dose of Compound 1 (200 mg, 400 mg, 700mg, or 1000 mg) or placebo. As shown in FIG. 12, healthy volunteers whoreceived Compound 1 experienced a decrease in blood 2,3-DPG levels at 24hours post-dose, relative to subjects who received the placebo.

In the MAD cohorts, the subjects' blood 2,3-DPG levels were measuredperiodically after dosing by a qualified LC-MS/MS method for thequantitation of 2,3-DPG in blood. The maximum decrease in 2,3-DPG on Day14 was 55% from baseline (median). 2,3-DPG levels reached a nadir andplateaued on Day 1 and had not returned to baseline levels 72 hoursafter the final dose on Day 14. Table 13 reports the median percentagechange in 2,3-DPG blood levels, relative to baseline, measured over timeafter the first dose on days 1 and 14 in healthy volunteers who receiveddaily doses of Compound 1 (100 mg BID, 200 mg BID, or 300 mg BID) orplacebo for 14 days. Accordingly, the median reduction in theconcentration of 2,3-DPG, relative to baseline, was at least about 25%at all dose levels tested 24 hours after administration of the firstdose on day 1 and at least about 40% at all dose levels tested 24 hoursafter administration of the first dose on day 14.

TABLE 13 Median Percentage Change in 2,3-DPG Levels (Days 1 and 14) DoseTime After 100 mg BID 200 mg BID 300 mg BID Placebo First Daily Day DayDay Day Dose 1 14 1 14 1 14 1 14 0 0.0 −42.0 0.0 −48.2 0.0 −59.4 0.0−7.6 6 −16.1 −44.3 −13.1 −48.5 −18.8 −53.0 −2.9 −10.9 8 −12.1 −44.7−22.3 −44.3 −23.8 −54.2 −0.6 −1.6 12 −18.1 −43.6 −23.1 −42.2 −31.6 −55.3−7.1 −1.6 16 −18.4 −43.9 −33.9 −42.9 −40.7 −52.4 −6.7 −5.3 24 −27.8−44.1 −43.5 −44.3 −50.8 −52.1 1.1 −10.7 48 −34.7 −38.7 −44.5 −1.0 72−20.2 −20.2 −32.9 −7.0

FIG. 13 is a graph of the blood 2,3-DPG levels measured over time inhealthy volunteers who received daily doses of Compound 1 (100 mg BID,200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shownin FIG. 13, healthy volunteers who received Compound 1 experienced adecrease in blood 2,3-DPG levels, relative subjects who received theplacebo. FIG. 14 is a graph of the blood 2,3-DPG levels measured on day14 in healthy volunteers who received daily doses of Compound 1 (100 mgBID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. Asshown in FIG. 14, healthy volunteers who received Compound 1 experienceda decrease in blood 2,3-DPG levels, relative to subjects who receivedthe placebo.

In the MAD cohorts, the subjects' blood ATP levels were measured on day14 by a qualified LC-MS/MS method for the quantitation of ATP in blood.ATP levels were elevated, relative to baseline, on day 14, and remainedelevated 60 hours after the last dose. Table 14 reports the medianpercentage change in blood ATP levels, relative to baseline, measuredover time after the first dose on day 14 in healthy volunteers whoreceived daily doses of Compound 1 (100 mg BID, or 200 mg BID) orplacebo for 14 days.

TABLE 14 Median Percentage Change in ATP Levels (Day 14) Time AfterFirst Dose Daily Dose 100 mg BID 200 mg BID Placebo 0 41.5 55.3 −0.5 643.8 48.1 2.8 8 47.8 58.4 −4.1 12 45.4 56.2 2.3 16 44.8 57.0 −6.8 2455.0 64.0 2.9 48 52.2 58.9 4.7 72 49.2 54.0 2.2

FIG. 15 is a graph of the blood ATP levels measured on day 14 in healthyvolunteers who received daily doses of Compound 1 (100 mg BID, 200 mgBID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shown in FIG.15, healthy volunteers who received Compound 1 experienced an increasein blood ATP levels, relative to subjects who received the placebo.

FIG. 16 is a graph plotting the blood concentration of Compound 1(ng/mL) measured in healthy volunteer (HV) patients on a first (left)axis and the concentration of 2,3-DPG (micrograms/mL) measured in theseHV patients on a second (right) axis after administration of a singledose of Compound 1 (400 mg). Solid symbols represent geometric means andStandard errors of the observed Compound 1 plasma and 2,3 DPGconcentrations. As shown in the figure, the observed 2,3 DPG modulationdoes not track directly plasma pharmacokinetics (blood concentration ofCompound 1) where the pharmacodynamic maximum (i.e., the minimum of the2,3-DPG concentration, at time ˜24 h) occurred nearly 24 h after thepharmacokinetic maximum (i.e., maximum of the PK curve, at time ˜1-2 h).The observed pharmacodynamic response in HVs was durable, with acalculated PD half-life of ˜20 h, where 2,3-DPG depression was observedlong after plasma levels were undetectable. Taken together, thissuggests that identifying the pharmacologically active dose cannot beadequately performed using pharmacokinetic parameters(C_(max)/C_(min)/AUC) in isolation, but rather support an approach thatincludes integrating the temporal pharmacokinetic/pharmacodynamicrelationship to provide the platform of evidence that QD dosing may befeasible in sickle cell disease patients.

Example 7: Analysis of ATP and 2,3 DPG in K2EDTA Whole Blood by LC-MS/MS

The following procedures are employed for the analysis of ATP and2,3-DPG in human whole blood K2EDTA using a protein precipitationextraction procedure and analysis by LC-MS/MS.

This bioanalytical method applies to the parameters described below:

Assay Range 25,000-1,500,000 ng/mL Extraction Volume 15.0 μLSpecies/Matrix/Anticoagulant Water as a surrogate for Human Whole BloodK2EDTA Extraction type Protein Precipitation Sample Storage 80° C. MassSpectrometer API-5500 Acquisition software Analyst/Aria System

The following precautions are followed:

1. Standard and QC samples are prepared on ice and stored in plasticcontainers.

2. Study samples and QC samples are thawed on ice.

3. Extraction is performed on ice.

The following definitions and abbreviations are employed:

CRB Carryover remediation blanks FT Freeze-thaw MPA Mobile phase A MPBMobile phase B NA Not applicable NR Needle rinse RT Retention time SIPStability in progress TBD To be determined

The following chemicals, matrix, and reagents are used:

K₂EDTA Human Whole Blood, BioreclamationIVT or equivalent (Note:BioReclamationIVT and BioIVT are considered equivalent) Acetonitrile(ACN), HPLC Grade or better Ammonium Acetate (NH₄OAc), HPLC grade orequivalent Ammonium Hydroxide (NH₄OH, 28-30%), ACS grade or betterDimethylsulfoxide (DMSO), ACS grade or better Formic Acid (FA), 88% ACSgrade Isopropanol (IPA), HPLC Grade or better Methanol (MeOH), HPLCGrade or better Water (H₂O), Milli-Q or HPLC Grade ATP-Analyte, Sponsoror supplier ATP-IS-IS, Sponsor or supplier 2,3-DPG-Analyte, Sponsor orsupplier 2,3-DPG-IS-IS, Sponsor or supplier

The following procedures are used for reagent preparation. Anyapplicable weights and volumes listed are nominal and may beproportionally adjusted as long as the targeted composition is achieved:

Nominal Volumes Final Solution for Solution Storage Solution CompositionPreparation Conditions Mobile Phase A 10 mM Ammoniumn Weigh Ambient(MPA) Acetate in water pH approximately Temperature 8.5 770.8 mg ofAmmonium Acetate; add to a bottle with 1000 mL of water. Adjust pH to8.3-8.7 using Ammonium Hydroxide. Mobile Phase B 5:95 MPA:ACN Add 50.0mL of Ambient (MPB) MPA to 950 mL of Temperature CAN. Mix. Needle Rinse1 25:25:25:25:0.1 Add 500 mL of Ambient (NR1) (v:v:v:v:v) MeOH, 500 mLof Temperature MeOH:ACN:H2O: ACN, 500 mL of IPA:NH₄OH H₂O, 500 mL ofIPA, and 2 mL of NH₄OH. Mix. Needle Rings 2 90:10:0.1 (v:v:v) Add 2 mLof FA to Ambient (NR2) H₂0:MeOH:FA 200 mL of MeOH Temperature and 1800mL of H₂0. Mix.

Calibration standards are prepared using water as the matrix accordingto the table presented below. The indicated standard is prepared bydiluting the indicated spiking volume of stock solution with theindicated matrix volume.

Stock Spiking Matrix Final Final Calibration Conc. Vol. Vol. Vol. Conc.Standard Stock Solution (ng/mL) (mL) (mL) (mL) (ng/mL) STD-6 ATP Stock60,000,000 0.0100 0.380 0.400 1,500,000 2,3-DPG Stock 60,000,000 0.0100STD-5 STD-6 1,500,000 0.100 0.200 0.300 500,000 STD-4 STD-6 1,500,0000.0500 0.325 0.375 200,000 STD-3 STD-6 1,500,000 0.0250 0.350 0.375100,000 STD-2 STD-5 500,000 0.0500 0.450 0.500 50,000 STD-1 STD-5500,000 0.0250 0.475 0.500 25,000 Cond. STD-5 500,000 0.0250 0.975 1.0012,500

Quality control standards are prepared using water as the matrixaccording to the table presented below. The indicated quality controlstandard is prepared by diluting the indicated spiking volume of stocksolution with the indicated matrix volume.

Quality Stock Spiking Matrix Final Final Control Conc. Vol. Vol. Vol.Conc. Standard Stock Solution (ng/mL) (mL) (mL) (mL) (ng/mL) QC-High ATPStock 60,000,000 0.160 7.68 8.00 1,200,000 2,3-DPG Stock 60,000,0000.160 QC-Mid QC-High 1,200,000 1.50 4.50 6.00 300,000 QC-Low QC-Mid300,000 1.50 4.50 6.00 75,000

An internal standard spiking solution is prepared with a finalconcentration of 12,500 ng/mL ATP and 2,3-DPG by diluting stocksolutions of ATP and 2,3-DPG at concentrations of 1,000,000 ng/mL withwater. 0.200 mL each of the ATP and 2,3-DPG stock solutions are dilutedwith 15.6 mL of water to produce a final volume of 16.0 mL at a finalconcentration of 12,500 ng/mL of ATP and 2,3-DPG.

The following procedures are used for sample extraction prior toanalysis via LC-MS/MS. 15.0 μL of the calibration standards, qualitycontrols, matrix blanks, and samples are aliquoted into a 96-well plate.50.0 μL of the internal standard spiking solution is added to allsamples on the plate, with the exception of the matrix blank samples;50.0 μL of water is added to the matrix blank samples. Subsequently, 150μL of water is added to all samples on the plate. The plate is thencovered and agitated by vortex at high speed for ten minutes, afterwhich 750 μL of methanol is added to all samples on the plate. The plateis covered and agitated by vortex for approximately 1 minute. The plateis then centrifuged at approximately 3500 RPM at approximately 4° C. forfive minutes. After centrifugation, a liquid handler is used to transfer50 μL of each sample to a new 96-well plate, and 200 μL of acetonitrileis added to all samples on the plate. The newly prepared plate iscovered and agitated by vortex for approximately 1 minute. The plate isthen centrifuged at approximately 3500 RPM at approximately 4° C. for 2minutes.

The following LC parameters and gradient conditions are used foranalysis of the extracted samples:

LC Parameters Analytical Column Vendor: SeQuant Description: ZIC-pHILICDimensions: 50 mm × 2.1 mm Column Heater Temperature: 40° C. Plate RackPosition: Cold Stack Cold Stack Set Point:  5° C. Mobile Phase MobilePhase A 10 mM Ammoniumn (MPA) Acetate in water pH 8.5 Mobile Phase B5:95 MPA:ACN (MPB) Injection Volume 5 μL

LC Gradient Time Flow Gradient Step (s) (mL/min) Setting % MPB 1 500.400 Step 5 2 30 0.400 Ramp 95 3 70 0.400 Step 5Data is collected starting at 0.08 min and is collected over a datawindow length of 0.70 min.

The following MS parameters are used for analysis of the extractedsamples using an API-5500 Mass Spectrometer:

Interface: Turbo Ion Spray Ionization, positive-ion mode Scan Mode:Multiple Reaction Monitoring (MRM) Scan Parameters: Parent/Product:Dwell Time (ms): 506.0/159.0 50 521.0/159.0 25 265.0/166.8 50268.0/169.8 25 Source Temperature: 400° C.

The collected MS data is analyzed and sample concentrations arequantified using peak area ratios with a linear 1/x² regression type.

We claim:
 1. A compound1-(5-((4-(difluoromethoxy)phenyl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2,2-dimethylpropan-1-one,or a pharmaceutically acceptable salt thereof.
 2. A compound1-(5-((4-(difluoromethoxy)phenyl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2,2-dimethylpropan-1-one.3. A pharmaceutical composition comprising the compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 4. A pharmaceutical composition comprising thecompound of claim 2 and a pharmaceutically acceptable carrier.
 5. Acomposition comprising the compound of claim 2, obtained by a processcomprising treating intermediate compound2-((4-(difluoromethoxy)phenyl)sulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrolewith 3-hydroxy-2,2-dimethylpropanoic acid in the presence of an amidecoupling reagent to afford the compound of claim 2.